Following primary herpes simplex virus type-1 (HSV-1) infection of the eye, virus replicates in the eye. Eye disease can result from direct damage inflicted on the corneal tissue by HSV-1, or indirectly, due to cytokines and chemokines released from cells infiltrating the cornea after infection. Virus is completely cleared between days 7-10 following ocular infection. The longer it takes for the immune system to clear the virus from the eye; the more extensive and protracted is the consequent ocular disease. Decreased virus load in the eye and accelerated virus clearance are probably the most efficient way to decrease latency and thus subsequent recurrent infections and loss of vision. Therefore, our main goal is to determine immune responses that are most efficacious at decreasing HSV-1 replication in the eye, reducing the establishment of latency, and reactivation from latency. We have shown recently that: (1) Immunization with a cocktail of 5 HSV-1 glycoproteins (5gP) (gB, gC, gD, gE, gl) is more efficacious than immunization with any single glycoprotein, gB+gD, or live HSV-1 vaccine; and (2) Immunization with 5gP DNA ("naked" DNA corresponding to the 5gP proteins) appeared even more efficacious than 5gP protein (subunit) immunization, resulting in further reduction of virus titers in the eye, more rapid viral clearance, lower establishment of latency in trigeminal ganglia (TGs), higher IL-12, and higher IFN-gamma (Appendix 1). Most recently, we have shown that DNA immunization also stimulates NK cells, macrophages and produces significantly higher numbers of CD11c+ and CD8 + T cells in the cornea of DNA-immunized mice than protein-immunized mice. We hypothesize that following DNA immunization signaling through dendritic cells (DCs) is important to the enhancement and maintenance of the production of CD8+/NK cells in the cornea of DNA-immunized mice. We further hypothesize that following DNA immunization signaling through macrophages (IL-12 production) is important in to the provision of the CD8+/NK cells with the signaling required to enhance and maintain IFN-gamma, production. Thus, to further elucidate the biologic and immunologic mechanisms responsible for reduced virus replication in the eye, more rapid clearance of HSV-1, and reduce latency in mice immunized with 5gP DNA, we propose to accomplish the following Specific Aims: 1. Confirm the hypothesis that the higher efficacy of 5gP DNA immunization is due to stimulation of both innate and adaptive immune responses, whereas the relatively lower efficacy of 5gP protein (the most effective protein vaccine against ocular HSV-1) immunization is mostly due to CD4-related responses. 2. Confirm the hypothesis that further stimulation of professional APCs using IL-12 and FLt3L as adjuvants will further enhance the activities of CD8 + T cells and NK cells leading to a greater reduction in ocular virus replication, latency, and reactivation in DNA-vaccinated mice.